Recycling System for Manipulation of Intracellular NADH Availability

ABSTRACT

The present invention describes a novel recombinant NADH recycling system that is used as a process for producing reduced compounds. In a specific embodiment, the reduced compounds include ethanol, succinate, lactate, a vitamin, a pharmaceutical and a biodegraded organic molecule. The NADH recycling system effects metabolic flux of reductive pathways in aerobic and anaerobic environments.

This application claims priority to U.S. Provisional Application Ser.No. 60/335,371, filed Nov. 2, 2001, which is incorporated by referenceherein in its entirety.

GOVERNMENT INTEREST

The present invention was developed with funds from the United StatesGovernment. Therefore, the United States Government may have certainrights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the fields of microbiology, molecularbiology, cell biology and biochemistry. More specifically, the presentinvention relates to manipulating reductive metabolic processes in vivousing genetic and metabolic engineering, thereby allowing externalcontrol of intracellular nicotinamide adenine dinucleotide (NADH)availability. Further, the present invention relates to a method ofproducing increased reduced metabolites such as ethanol through aerobicor anaerobic growth of a living system comprised of a recombinant NADHrecycling system.

2. Related Art

The metabolic pathways leading to the production of most industriallyimportant compounds involve oxidation-reduction (redox) reactions.Biosynthetic transformations involving redox reactions offer asignificant economic and environmental advantage for the production offine chemicals over conventional chemical processes, in particular thoseredox reactions requiring stereospecificity. Furthermore, biodegradationof toxic chemicals often also involves redox reactions.

Nicotinamide adenine dinucleotide (NAD) functions as a cofactor in over300 redox reactions and regulates various enzymes and genetic processes(Foster et al., 1990). The NADH/NAD+ cofactor pair plays a major role inmicrobial catabolism in which a carbon source, such as glucose, isoxidized using NAD+ producing reducing equivalents in the form of NADH.It is crucially important for continued cell growth that this reducedNADH be oxidized to NAD+ and a redox balance be achieved. Under aerobicgrowth, oxygen achieves this recycling by acting as the oxidizing agent.While under anaerobic growth, and in the absence of an alternateoxidizing agent, the regeneration of NAD+ is achieved throughfermentation by using NADH to reduce metabolic intermediates.

The metabolic pathways leading to the production of most industriallyimportant compounds involve redox reactions. Biosynthetictransformations involving redox reactions also offer a considerablepotential for the production of fine chemicals over conventionalchemical processes, especially those requiring stereospecificity.

Enzymes referred to in general as oxidoreductases, or more specificallyas oxidases, reductases or dehydrogenases, catalyze these biologicalredox reactions. These enzymes require a donor and/or an acceptor ofreducing equivalents in the form of electrons, hydrogen or oxygen atoms.Cofactor pairs that are transformed reversibly between their reduced andoxidized states, nucleotide cofactors such as NADH/NAD+ and NADPH/NADP+among others, serve as donors and/or acceptors of reducing equivalentsvery effectively in a living cell.

The NADH/NAD+ cofactor pair has demonstrated a regulatory effect on geneexpression and enzymatic activity. Examples include, among others, theinduction by NADH of adhE expression, which encodes an alcoholdehydrogenase (Leonardo et al., 1993; Leonardo et al., 1996) andcatalyzes the production of ethanol during fermentation, the inhibitionby high NADH/NAD+ ratios on the pyruvate dehydrogenase complex (Graef etal., 1999), and the regulation by the NADH/NAD+ ratio on the shiftbetween oxidation or reduction of L-lactaldehyde (Baldoma and Aguilar,1988).

The ratio of the reduced to oxidized form of this cofactor, theNADH/NAD+ ratio, is critical for the cell. The NAD(H/+) cofactor pair isvery important in microbial catabolism, where a carbon source, such asglucose, is oxidized through a series of reactions utilizing NAD+ as acofactor and producing reducing equivalents in the form of NADH. It iscrucially important for the continued growth of the cell that thisreduced NADH be oxidized to NAD+, thus achieving a redox balance. Underaerobic growth, oxygen achieves this by acting as the oxidizing agent.While under anaerobic growth and in the absence of an alternateoxidizing agent, this process occurs through fermentation, where NADH isused to reduce metabolic intermediates and regenerate NAD+ (FIG. 1).

The high influence of cofactors in metabolic networks has been evidencedby studies in which the NADH/NAD+ ratio has been altered by feedingcarbon sources possessing different oxidation states (Alam and Clark,1989; Leonardo et al., 1996), by supplementing anaerobic growth withdifferent electron acceptors, such as fumarate and nitrate (Graef etal., 1999) and by expressing an enzyme like NADH oxidase (Lopez deFelipe et al., 1998). Other previous efforts to manipulate NADH levelshave included the addition of electron dye carriers (Park and Zeikus,1999) and the variation of oxidoreduction potential conditions (Riondetet al., 2000).

The effective regeneration of used cofactors is critical in industrialcofactor-dependent production systems because of the impeding high costof cofactors such as NAD. The cofactors, also referred to as co-enzymes,NAD+ and NADP+ are expensive chemicals, thereby making theirregeneration by reoxidation to the original state imperative if they areto be used economically in low cost, chemical production systems.Efforts to do such have been described. U.S. Pat. No. 4,766,071describes in vitro regeneration of NADH using a cell lysate ofClostridium kluyveri as a biocatalyst and an aldehyde as an oxidizingagent. U.S. Pat. No. 5,393,615 describes electrochemical regeneration ofNADH using an electrode characterized by a mediator function. Similarly,U.S. Pat. No. 5,264,092 discloses mediators covalently attached to apolymeric backbone wherein the polymeric backbone coats the surface ofan electrode. U.S. Pat. No. 5,302,520 discloses a NAD regenerationsystem and an adenosine phosphate regeneration system that, in thepresence of pyruvate, yields a labeled carbohydrate.

In enzyme bioreactors, NAD+-dependent formate dehydrogenase (FDH) frommethylotrophic yeast and bacteria is extensively used to regenerate NADHfrom NAD+ in vitro. FDH catalyzes the practically irreversible oxidationof formate to CO₂ and the simultaneous reduction of NAD+ to NADH. Thissystem of cofactor regeneration has been successfully applied in theproduction of optically active amino acids (Galkin et al., 1997), chiralhydroxy acids, esters, alcohols, and other fine chemicals synthesized bydifferent dehydrogenases (Hummel and Kula, 1989), (Tishkov et al.,1999). Purified FDH has also been used to regenerate NADH in vitro forthe industrial production of non-natural amino acids that cannot beobtained by fermentation, such as L-tert-leucine which has importantapplications when used in pharmaceuticals (Kragl et al., 1996).

In spite of these advances, biotransformation with whole cells remainsthe preferred industrial method for the synthesis of mostcofactor-dependent products. In these systems, the cell naturallyregenerates the cofactor; however, the enzyme of interest has to competefor the required cofactor with a large number of other enzymes withinthe cell. For this reason, in cofactor-dependent production systemsutilizing whole cells, after the enzymes of interest have beenoverexpressed, cofactor levels and the availability of the required formof the cofactor (reduced or oxidized) become crucial for optimalproduction.

Furthermore, one of the long-sought goals in recombinant polypeptideproduction processes is to achieve a high cloned gene expression leveland high cell density. Unfortunately, under these demanding conditions,the amount of acetate accumulated in the reactor increasesprecipitously. Acetate accumulation is associated with decreasedrecombinant polypeptide productivity (Aristidou et al., 1995). Methodsof controlling acetate production would be beneficial in increasingrecombinant polypeptide yield in large-scale industrial synthesis ofpolypeptides. Additionally, the sort of metabolic manipulation used toincrease recombinant polypeptide yields could also he applied to theproduction of any biomolecule in a large-scale system in which thestress of biomolecule production normally leads to acetate accumulation,such as biopolymers.

Catalytic hydrodesulfurization has the potential to remove sulfur fromvarious fuels. However, this technology is associated with high costsdue to hydrogen consumption and heavy metal deactivation of thecatalyst. A lower cost treatment is microbiological biodesulfurization.U.S. Pat. No. 6,337,204 describes a Rhodococcus bacterial culturecapable of biodesulfurization. One obstacle in this method is that thesereactions require NADH as a cofactor, the availability of which is alimiting factor.

Although it is generally known that cofactors play a major role in theproduction of different fermentation products, their role has not beenstudied thoroughly and systematically in engineered systems. Instead,metabolic engineering studies have focused on manipulating enzyme levelsthrough the amplification, addition or deletion of a particular pathway.Such steps relegate cofactor manipulations as a powerful tool formetabolic engineering, as many enzymes require them. The dehydrogenasesare but one example of selective catalysis requiring theenergy-transferring redox couple, NADH/NAD+.

Prior to the present invention, a genetic means of manipulating theavailability of intracellular NADH in vivo by regenerating NADH throughthe heterologous expression of an NAD+-dependent formate dehydrogenasewas not known. By way of the present invention, the effect ofmanipulating intracellular NADH on the metabolic patterns in Escherichiacoli under anaerobic and aerobic conditions by substituting the nativecofactor-independent formate dehydrogenase (FDH) by an NAD+-dependentFDH such as from Candida boidinii is described. This manipulationprovoked a significant change in the final metabolite concentrationpattern both anaerobically and aerobically. Under anaerobic conditions,the production of more reduced metabolites was favored, as evidenced bya dramatic increase in the ethanol to acetate ratio. Unexpectedly duringaerobic growth, the increased availability of NADH induced a shift tofermentation even in the presence of oxygen by stimulating pathways thatare normally inactive under these conditions.

SUMMARY OF THE INVENTION

The present invention is directed to a method for increasing theintracellular availability of NADH, comprising the transformation of acell with a nucleic acid encoding an NAD+-dependent dehydrogenase andgrowth of said cell under conditions in which said NAD+-dependentdehydrogenase increases the intracellular availability of NADH. In aspecific embodiment, the NAD+-dependent dehydrogcnase is a formatedehydrogenase. In a further specific embodiment, the formatedehydrogenase is Candida boidinii formate dehydrogenase.

The present invention is directed to methods of utilizing a recombinantNADH recycling system to produce NADH and other metabolites in vivo. Oneembodiment of the present invention is a method to produce NADH in vivocomprising growing a culture of cells that comprises at least one cell,comprising a recombinant NADH recycling system. In a specific embodimentof the invention, the cell which comprises the recombinant NADHrecycling system is a bacterium, including E. coli. In further specificembodiments of the invention, the recombinant NADH recycling systemcomprises a nucleotide sequence encoding a NAD+-dependent formatedehydrogenase, which may be from, but is not limited to, yeast, orCandida boidinii, operatively linked to a promoter

In another embodiment of the present invention, there is a cellcomprising a recombinant NADH recycling system. In specific embodiments,the recombinant NADH recycling system of the cell comprises a nucleotidesequence encoding a NAD+-dependent formate dehydrogenase operativelylinked to a promoter. In a specific embodiment, the sequence isheterologous.

Yet another embodiment of the invention is a method to produce a reducedcompound in vivo comprising growing a culture of cells that comprises atleast one cell comprising a recombinant NADH recycling system. In aspecific embodiment, the reduced compound produced is ethanol, lactate,succinate, a vitamin, a pharmaceutical or a biodegraded organicmolecule. In a further specific embodiment, the pharmaceutical compoundis an antibiotic. In another specific embodiment, the growing of cellculture takes place in an oxygen-deficient atmosphere. In anotherspecific embodiment, the growing is in an oxygen-rich atmosphere. Inanother specific embodiment, formate is added to the culture of cells.In a further specific embodiment, the amount of formate added is atleast about 100 mM.

In an additional embodiment of the present invention there is a methodto produce ethanol comprising growing a culture of cells wherein theculture comprises at least one cell comprising a recombinant NADHrecycling system.

Yet another embodiment of the invention is a method of alteringmetabolic flux of a reduction pathway comprising growing a culture ofcells, wherein the culture comprises at least one cell comprising arecombinant NADH recycling system, and the flux of the metabolic pathwayis redistributed as compared to a normal metabolic flux of the pathway.

Another embodiment of the invention is a method of biodegradation invivo comprising growing a culture of cells, wherein the culturecomprises at least one cell comprising a recombinant NADH recyclingsystem.

Yet another embodiment of the present invention is a method forbiodesulfurization in vivo comprising growing a culture of cells,wherein the culture comprises at least one cell comprising a recombinantNADH recycling system. In a further embodiment, the cells are bacteriacells. In a specific embodiment, the bacterial cells are Rhodococcusbacteria.

Another embodiment of the present invention is a method of biopolymerproduction in vivo comprising growing a culture of cells, wherein theculture comprises at least one cell comprising a recombinant NADHrecycling system.

One embodiment of the present invention is a method for polypeptideproduction in vivo comprising growing a culture of cells, wherein theculture comprises at least one cell comprising a recombinant NADHrecycling system. A specific embodiment is the production ofheterologous recombinant protein.

Other embodiments, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF SUMMARY OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein:

FIG. 1. Schematic representation of Escherichia coli central anaerobicmetabolic pathways illustrating involvement of the NADH/NAD+ cofactorpair.

FIG. 2. Diagram illustrating the native cofactor-independent formatedegradation pathway and the newly introduced NAD+-dependent pathway.

FIG. 3A to 3F. Graphical illustrations of results of anaerobic tubeexperiments of strains after 72 hours.

FIGS. 4A and 4B. Graphical illustrations of (A) formate consumed andethanol/acetate ratio (B) of strains grown in anaerobic tubeexperiments.

FIG. 5A to 5F. Graphical illustrations of aerobic shake flask experimentafter 24 hours.

FIGS. 6A and 6B. Graphical illustrations of (A) lactate and (B)succinate concentrations from aerobic growth in various concentrationsof supplemented formate.

FIG. 7. Central aerobic metabolic pathway of Escherichia coli showinggeneration of NADH and regeneration of NAD+ and metabolic flux of eachcontributing pathway.

FIG. 8. Diagram illustrating the native cofactor-independent formatedegradation pathway and the recombinant NADH recycling system.

FIG. 9. Diagram showing the uptake of 1 C-mole of glucose in a celltogether with yields of reduced products obtained in an anaerobicchemostatic experiment.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more.

As used herein, the expressions “cell”, “cell line” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

As used herein, the term “recombinant” cells or host cells are intendedto refer to a cell into which an exogenous nucleic acid sequence, suchas, for example, a vector, has been introduced. Therefore, recombinantcells are distinguishable from naturally occurring cells which do notcontain a recombinantly introduced nucleic acid. Recombinant DNA refersto DNA which has been modified by joining genetic material from twodifferent sources, which may be different species or the same species.Recombinant polypeptides may be the gene products of recombinant DNA, orpolypeptides produced in recombinant cells. The term “recombinant NADHrecycling system” refers to an engineered system for the recycling ofNADH. It can refer to cells that comprise this system, or recombinantDNA or polypeptide sequences that comprise such a system.

The terms “modified” or “modification” as used herein refer to the stateof a metabolic pathway being altered in which a step or process in thepathway is increased or upregulated, such as in activity of an enzyme orexpression of a nucleic acid sequence, respectively. In a specificembodiment, the modification is the result of an alteration in a nucleicacid sequence which encodes an enzyme in the pathway, an alteration inexpression of a nucleic acid sequence which encodes an enzyme in thepathway, or an alteration in translation or proteolysis of an enzyme inthe pathway (i.e., formate dehydrogenase), or a combination thereof. Askilled artisan recognizes that there are commonly used standard methodsin the art to obtain the alterations, such as by mutation.

Nucleic acid is “operatively linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operatively linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operativelylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operatively linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operatively linked” means that the DNA sequences being linked arecontiguous and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, then synthetic oligonucleotide adaptors or linkersare used in accord with conventional practice.

“Plasmids” are designated by lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein arecommercially available, are publicly available on an unrestricted basis,or can be constructed from such available plasmids in accord withpublished procedures. In addition, other equivalent plasmids are knownin the art and will be apparent to the ordinary artisan.

One embodiment of the present invention is to provide a method toproduce NADH in vivo comprising growing a culture of cells thatcomprises at least one cell comprising a recombinant NADH recyclingsystem, under conditions to produce NADH. The NADH recycling systemcomprises a nucleic acid sequence encoding a dehydrogenase that isNAD+-dependent formate, such as FDH. The recombinant NADH recyclingsystem increases the intracellular availability of NADH. FDH catalyzesthe practically irreversible oxidation of formate to CO₂ and thesimultaneous reduction of NAD+ to NADH. A skilled artisan is aware thatNADH and NAD+ refer to nicotinamide adenine dinucleotide in two distinctoxidation states, respectively, and are cofactors that mediate a largenumber of biological oxidations and reductions, which generally providea step or steps in catabolic metabolic pathways. When a substrate ishydrolyzed (in the instant case, formate is the substrate) hydride (H⁻)is transferred to the C-4 of the nicotinamide ring of NAD+, and the H⁺is lost to the medium. Further, a skilled artisan recognizes that adehydrogenase such as formate dehydrogenase catalyzes a reversiblehydride transfer, generally a stereospecific reaction owing to the twodistinct domains that the dehydrogenase protein conforms, in which eachdomain is specific for the binding of cofactor or substrate.

A skilled artisan recognizes that sequences useful in the presentinvention may be obtained in a database such as the National Center forBiotechnology's GenBank database. For example, a NAD+-dependent FDH1 ofCandida boidinii (SEQ ID NO: 1, GenBank Accession NO: AF004096) and is anon-limiting example of a suitable FDH of the present invention. SEQ IDNO: 1 and any other nucleotide sequences encoding the polypeptide SEQ IDNO: 2 (GenBank Accession NO: AAC49766) are suitable. Other suitable FDHsare from Candida methylica (SEQ ID NO: 3, GenBank Accession NO:CAA57036), Pseodomonas sp. 101 (SEQ ID NO: 4, GenBank Accession NO:P33160), Arabidopsis thaliana (SEQ ID NO: 5, GenBank Accession NO:AAF19436), and Staphylococcus aureus (SEQ ID NO: 6, GenBank AccessionNO: BAB94016). Any nucleic acids encoding SEQ ID NOS: 3, 4, 5, and 6 arealso appropriate. Other species in embodiments of the invention arecontemplated, such as Saccharomyces bayanus, Saccharomyces exiguus,Saccharomyces servazzii, Zygosaccharomyces rouxii, Saccharomyceskluyveri, Kluyveromyces thermotolerans, Kluyveromyces lactis,Kluyveromyces marxianus, Pichia angusta, Debaryomyces hansenii, Pichiasorbitophila, Candida tropicalis and Yarrowia lipolytica. Standardmethods and reagents in the field of molecular biology are well known inthe art. A reference for such methods includes Current Protocols inMolecular Biology, Chapter 13 (Ausubel et al., 1994), hereinincorporated by reference.

In a preferred embodiment, the nucleic acid sequence encoding thenon-native FDH is inserted into a vector such that the expression of thenon-native FDH is controlled by a promoter that is operatively linked tothe non-native FDH. A skilled artisan is aware of appropriate vectorsand promoters, not excluding the native promoter of the non-native FDHgene, for expression of a recombinant gene in a host organism andmethods to develop a resulting recombinant plasmid. The recombinantplasmid comprising the non-native FDH and promoter are then transformedinto a host cell by methods well known in the art.

In a specific embodiment the host organism is an anaerobe, such as, forexample, Escherichia coli, a facultative anaerobe that grows either inthe presence or the absence of oxygen, or any aerotolerant organism thatis capable of fermentation. In another specific embodiment, thewild-type FDH gene is inactivated, meaning that the nucleic acidsequence encoding for the native FDH gene is inoperative. For example,the fdhF (SEQ ID NO:7, GenBank Accession NO: M13563) of E. coli isreplaced by homologous recombination using methods well known in theart. Engineering a host cell such that an enzymatic activity is removedcan be screened, in one manner, by confirming the lack of the enzymaticactivity in the recombinant host cell. Upon expression of the plasmid,the recombinant FDH gene assumes the responsibility of providing therespective enzymatic activity (e.g. dehydrogenation) for the host cell.

For example, the nucleic acid sequence of the formate dehydrogenase isregulated by an inducible promoter.

The recombinant cell is grown in an oxygen-rich atmosphere (aerobic) orin an oxygen-deficient atmosphere (anaerobic) to produce NADH, therebyincreasing intracellular availability of NADH.

By increasing intracellular NADH availability, the present inventionprovides a method to produce a reduced metabolite comprising growing aculture of cells that comprises at least one cell comprising arecombinant NADH recycling system, and removing the reduced metabolitefrom the culture. The NADH recycling system effects increasedintracellular NADH availability as compared to a control cell andconsequently accumulates reduced products and metabolites such as, forexample, reduced metabolites of glucose.

The method to produce reduced metabolites of the present inventionincludes metabolites not originally synthesized by the host cell. Forexample, in vivo reduction provided by the present invention is appliedto biodegradation of toxic chemicals and/or semi-synthesis of acompound, wherein the compound is, for example, a vitamin or apharmaceutical or a medicament. The pharmaceutical compound may be anantibiotic, such as tetracycline, amoxicillin, erythromycin, orzithromycin. Often the syntheses of such compounds to an appropriateoxidation state is required to, for example, ensure solubility, and suchrequirements include a reduction reaction. In such cases, the method ofthe present invention is contemplated especially involving a reductionreaction where stereospecificity is desired.

In yet another object of the present invention is a method to produceethanol comprising growing a culture of cells that comprises at leastone cell comprising a recombinant NADH recycling system, and removingthe ethanol from the culture.

Another object is a method to produce lactate comprising growing aculture of cells, wherein the culture comprises at least one cellcomprising a NADH recycling system, wherein the lactate may be removedfrom the culture.

It is another object of the present invention to provide a method toproduce succinate comprising growing a culture of cells, wherein theculture comprises at least one cell comprising a NADH recycling system,wherein the succinate may be removed from the culture.

One object is a method of altering metabolic flux of a metabolic pathwaycomprising growing a culture of cells, wherein the culture comprises atleast one cell comprising a NADH recycling system. The present inventionenables the altering of metabolic flux of a reduction pathway to producea reduced metabolite or reduced compound.

In a specific embodiment, the compound to be degraded is anenvironmental toxin, such as a toxic organic or inorganic compound. Askilled artisan recognizes that a toxic organic pollutant (also referredto as a xenobiotic) includes but is not limited to benzene, toluene,ethylbenzene, o-xylene, m-xylene, p-xylene, phenol, o-cresol, m-cresol,p-cresol, or styrene, as well as halogenated organic compounds such aspentachlorophenol. Examples of other environmental toxins includepetroleum hydrocarbons (such as fuel oil or gasoline), insecticides(such as polychlorinated biphenyls (PCBs) or DDT), halogenatedhydrocarbons, chlorinated benzenes, chlorophenols, chloroquaiacols,chloroveratroles, chlorocatechols, chlorinated aliphatics, perchlorates,nitrates, hydrolysates, or polycyclic aromatic hydrocarbons (PAHs, suchas phenanthrene).

Another object of the present invention is a method ofbiodesulfurization in order to remove sulfur from fossil fuels, such ascrude oil. Such an embodiment comprises at least one cell comprising anNADH recycling system where the NADH produced is a necessary cofactorfor the enzymes involved in the biodesulfurization pathway.Dibenzothiophene is a model compound for organic sulfur in fossil fuels.Known members of the dibenzothiophene desulfurization pathway includedibenzothiophene monooxygenase, dibenzothipohene-5,5-dioxidemonooxygenase, and 2′-hydroxybiphenly-2-sulfinate sulfinoylase. Knownbacterial strains which are capable of breaking down dibenzothiopheneusing this pathway include Rhodococcus strains, including IGTS8, T09,and RA-18, and Gordonia desulfuricans 213E. Also capable ofbiodesulfurization are E. coli that express recombinant genes fromRhodococcus, and Pseudomonas putida that express recombinant genes fromRhodococcus. Gordonia rubropertinctus strain T08 is capable ofbiodesulfurization using a novel pathway. Rhodococcus strain IGTS8,Gordonia rubropertinctus strain T08, E. coli, and Pseudomonas putida areavailable from the American Type Culture Collection (ATCC). In oneembodiment, a cell or cells comprising the NADH recycling system aretransformed with vectors that are capable of expressing the geneproducts of the biodesulfurization pathway genes. In another embodiment,cells capable of biodesulfurization are transformed with a recombinantNADH recycling system. In such an embodiment, the cells capable ofbiodesulfurization may be Rhodococcus or recombinant E. coli.

It is an object of the present invention to create a method for theproduction of biopolymers in bacteria. Such an embodiment comprises atleast one cell comprising an NADH recycling system where the NADHproduced is a necessary cofactor for the enzymes involved in thebiopolymer production pathway. The enzymes involved in the biopolymerproduction pathway may be host cell enzymes or recombinant enzymes.Biopolymers are polymers that are either naturally occurring or can beproduced through engineering of a host organism. Examples of biopolymersare polysaccharides, polythioesters, polyhydroxybutyrates,polyhydroxyalkanoates. Other examples are chitins, starch, lignin,glycogen, cellulose, and xanthan gum. In some embodiments, biopolymerscan also include polypeptides and amino acid polymers.

Another object of the present invention is a method of producingpolypeptides in bacteria. The polypeptides produced may be under thetranscriptional control of the host cell, or may be encoded by nucleicacids operatively linked to a promoter. The polypeptide may be of hostcell origin or heterologous, and may be recombinant. Heterologous refersto polypeptides not naturally occurring in the host. Heterologouspeptides may be from another species. Heterologous polypeptides may beencoded for by heterologous nucleic acids. Such an embodiment comprisesat least one cell comprising an NADH recycling system where the NADHproduced is able to shift the metabolic pattern of the cell to causedecreased acetate levels. Decreased acetate levels are associated withincreased yields of recombinant polypeptide.

Nucleic Acid-Based Expression Systems

1. Vectors

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can he replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques, which are described in Maniatis et al., 1988 and Ausubel etal., 1994, both incorporated herein by reference.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operatively linked codingsequence in a particular host organism. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

a. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind such as RNA polymerase and other transcriptionfactors. The phrases “operatively positioned,” “operatively linked,”“under control,” and “under transcriptional control” mean that apromoter is in a correct functional location and/or orientation inrelation to a nucleic acid sequence to control transcriptionalinitiation and/or expression of that sequence. A promoter may or may notbe used in conjunction with an “enhancer,” which refers to a cis-actingregulatory sequence involved in the transcriptional activation of anucleic acid sequence. In specific embodiments, the promoter functionsin a prokaryotic cell.

A promoter may be one naturally associated with a gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other prokaryotic, viral, or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. In addition to producing nucleicacid sequences of promoters and enhancers synthetically, sequences maybe produced using recombinant cloning and/or nucleic acid amplificationtechnology, including PCR™, in connection with the compositionsdisclosed herein (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906,each incorporated herein by reference). Furthermore, it is contemplatedthe control sequences that direct transcription and/or expression ofsequences within non-nuclear organelles such as mitochondria,chloroplasts, and the like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in the celltype, organelle, and organism chosen for expression. Those of skill inthe art of molecular biology generally know the use of promoters,enhancers, and cell type combinations for protein expression, forexample, see Sambrook et al. (1989), incorporated herein by reference.The promoters employed may be constitutive, tissue-specific, inducible,and/or useful under the appropriate conditions to direct high levelexpression of the introduced DNA segment, such as is advantageous in thelarge-scale production of recombinant proteins and/or peptides. Thepromoter may be heterologous or endogenous.

b. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector. (See Carbonclli et al., 1999, Levenson et al., 1998,and Cocea, 1997, incorporated herein by reference.) “Restriction enzymedigestion” refers to catalytic cleavage of a nucleic acid molecule withan enzyme that functions only at specific locations in a nucleic acidmolecule. Many of these restriction enzymes are commercially available.Use of such enzymes is widely understood by those of skill in the art.Frequently, a vector is linearized or fragmented using a restrictionenzyme that cuts within the MCS to enable exogenous sequences to beligated to the vector. “Ligation” refers to the process of formingphosphodiester bonds between two nucleic acid fragments, which may ormay not be contiguous with each other. Techniques involving restrictionenzymes and ligation reactions are well known to those of skill in theart of recombinant technology.

c. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated. Inspecific embodiments, the origin of replication functions in aprokaryotic cell.

d. Selectable and Screenable Markers

In certain embodiments of the invention, the cells contain nucleic acidconstruct of the present invention, a cell may be identified in vitro orin vivo by including a marker in the expression vector. Such markerswould confer an identifiable change to the cell permitting easyidentification of cells containing the expression vector. Generally, aselectable marker is one that confers a property that allows forselection. A positive selectable marker is one in which the presence ofthe marker allows for its selection, while a negative selectable markeris one in which its presence prevents its selection. An example of apositive selectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Further examples ofselectable and screenable markers are well known to one of skill in theart.

2. Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these term also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organisms that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny.

Host cells may be prokaryotic, depending upon whether the desired resultis replication of the vector or expression of part or all of thevector-encoded nucleic acid sequences. Numerous cell lines and culturesare available for use as a host cell, and they can be obtained throughthe American Type Culture Collection (ATCC), which is an organizationthat serves as an archive for living cultures and genetic materials,which is readily accessible on the world wide web. An appropriate hostcan be determined by one of skill in the art based on the vectorbackbone and the desired result. A plasmid or cosmid, for example, canbe introduced into a prokaryote host cell for replication of manyvectors. Bacterial cells used as host cells for vector replicationand/or expression include DH5α, JM109, and KC8, as well as a number ofcommercially available bacterial hosts such as SURE® Competent Cells andSOLOPACK™ Gold Cells (STRATAGENE®, La Jolla). Alternatively, bacterialcells such as E. coli LE392 could be used as host cells for phageviruses.

Similarly, a viral vector may be used in conjunction with a prokaryotichost cell, particularly one that is permissive for replication orexpression of the vector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in prokaryotic cells. One of skill in the art wouldfurther understand the conditions under which to incubate all of theabove described host cells to maintain them and to permit replication ofa vector. Also understood and known are techniques and conditions thatwould allow large-scale production of vectors, as well as production ofthe nucleic acids encoded by vectors and their cognate polypeptides,proteins, or peptides.

Cells may be grown in culture medium. Culture medium may be liquid orsolid. Liquid culture medium may be a broth. Solid medium may be moldedinto a plate. Liquid media are used for growth of pure batch cultureswhile solidified media are used widely for the isolation of purecultures, for estimating viable bacterial populations, and a variety ofother purposes. The usual gelling agent for solid or semisolid medium isagar, a hydrocolloid derived from red algae. Agar is used because of itsunique physical properties (it melts at 100 degrees and remains liquiduntil cooled to 40 degrees, the temperature at which it gels) andbecause it cannot be metabolized by most bacteria. Hence as a mediumcomponent it is relatively inert; it simply holds (gels) nutrients thatare in aquaeous solution. Types of culture medium include differential,selective, minimal, and enrichment.

3. Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with the present invention to producenucleic acid sequences, or their cognate polypeptides, proteins andpeptides. Many such systems are commercially and widely available.

An expression system from STRATAGENE® (La Jolla, Calif.) is the pET E.COLI EXPRESSION SYSTEM is a widely used in vivo bacterial expressionsystem due to the strong selectivity of the bacteriophage T7 RNApolymerase, the high level of activity of the polymerase and the highefficiency of translation. One of skill in the art would know how toexpress a vector, such as an expression construct, to produce a nucleicacid sequence or its cognate polypeptide, protein, or peptide.

4. Derivatives of Promoter Sequences

One aspect of the invention provides derivatives of specific promoters.One means for preparing derivatives of such promoters comprisesintroducing mutations into the promoter sequences. Such mutants maypotentially have enhanced, reduced, or altered function relative to thenative sequence or alternatively, may be silent with regard to function.

Mutagenesis may be carried out at random and the mutagenized sequencesscreened for function. Alternatively, particular sequences which providethe promoter region with desirable expression characteristics could beidentified and these or similar sequences introduced into other relatedor non-related sequences via mutation. Similarly, non-essential elementsmay be deleted without significantly altering the function of thepromoter. It is further contemplated that one could mutagenize thesesequences in order to enhance their utility in expressing transgenes,especially in a gene therapy construct in humans.

The means for mutagenizing a DNA segment comprising a specific promotersequence are well-known to those of skill in the art. Mutagenesis may beperformed in accordance with any of the techniques known in the art,such as, and not limited to, synthesizing an oligonucleotide having oneor more mutations within the sequence of a particular regulatory region.In particular, site-specific mutagenesis is a technique useful in thepreparation of promoter mutants, through specific mutagenesis of theunderlying DNA. The technique further provides a ready ability toprepare and test sequence variants, by introducing one or morenucleotide sequence changes into the DNA.

Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 toabout 75 nucleotides or more in length is preferred, with about 10 toabout 25 or more residues on both sides of the junction of the sequencebeing altered.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by various publications. As will be appreciated,the technique typically employs a phage vector which exists in both asingle stranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage are readily commercially available and their use is generally wellknown to those skilled in the art. Double stranded plasmids also areroutinely employed in site directed mutagenesis to eliminate the step oftransferring the gene of interest from a plasmid to a phage.

Alternatively, the use of PCR™ with commercially available thermostableenzymes such as Taq polymerase may be used to incorporate a mutagenicoligonucleotide primer into an amplified DNA fragment that can then becloned into an appropriate cloning or expression vector. ThePCR™-mediated mutagenesis procedures of Tomic et al. (1990) and Upenderet al. (1995) provide two examples of such protocols.

The preparation of sequence variants of the selected promoter orintron-encoding DNA segments using site-directed mutagenesis is providedas a means of producing potentially useful species and is not meant tobe limiting as there are other ways in which sequence variants of DNAsequences may be obtained. For example, recombinant vectors encoding thedesired promoter sequence may be treated with mutagenic agents, such ashydroxylamine, to obtain sequence variants.

Typically, vector mediated methodologies involve the introduction of thenucleic acid fragment into a DNA or RNA vector, the clonal amplificationof the vector, and the recovery of the amplified nucleic acid fragment.Examples of such methodologies are provided by U.S. Pat. No. 4,237,224,incorporated herein by reference. A number of template dependentprocesses are available to amplify the target sequences of interestpresent in a sample, such methods being well known in the art andspecifically disclosed herein.

One efficient, targeted means for preparing mutagenized promoters orenhancers relies upon the identification of putative regulatory elementswithin the target sequence. These can be identified, for example, bycomparison with known promoter sequences. Sequences which are sharedamong genes with similar functions or expression patterns are likelycandidates for the binding of transcription factors and are likelyelements to confer tissue specific expression patterns.

Other assays may be used to identify responsive elements in a promoterregion or gene. Such assays will be known to those of skill in the art(see for example, Sambrook et al., 1989; Zhang et al, 1997; Shan et al.,1997; Dai and Burnstein, 1996; Cleutjens et al., 1997; Ng et al., 1994;Shida et al., 1993), and include DNase I footprinting studies,Elecromobility Shift Assay patterns (EMSA), the binding pattern ofpurified transcription factors, effects of specific transcription factorantibodies in inhibiting the binding of a transcription factor to aputative responsive element, Western analysis, nuclear run-on assays,and DNA methylation interference analysis.

Preferred promoter constructs may be identified that retain the desired,or even enhanced, activity. The smallest segment required for activitymay be identified through comparison of the selected deletion ormutation constructs. Once identified, such segments may be duplicated,mutated, or combined with other known or regulatory elements and assayedfor activity or regulatory properties. Promoter region sequences used toidentify regulatory elements can also be used to identify and isolatetranscription factors that bind a putative regulatory sequence orelement, according to standard methods of protein purification, such asaffinity chromatography, as discussed above.

Preferably, identified promoter region sequences, whether used alone orcombined with additional promoters, enhancers, or regulatory elements,will be induced and/or regulated by an external agent, such as ahormone, transcription factor, enzyme, or pharmaceutical agent, toexpress operatively linked genes or sequences (Zhang et al., 1997; Shanet al., 1997). Alternatively, such a construct may be designed to ceaseexpression upon exposure to an external agent.

Following selection of a range of deletion mutants of varying size, theactivities of the deleted promoters for expression of the linked CATgene may be determined according to standard protocols.

The precise nature of the deleted portion of the promoter may bedetermined using standard DNA sequencing, such as Sanger dideoxytermination sequencing, to identify which promoter sequences have beenremoved in each of the assayed deletion mutants. Thus, a correlation maybe obtained between the presence or absence of specific elements withinthe promoter sequence and changes in activity of the linked reportergene.

5. FDH Nucleic Acids

a. Nucleic Acids and Uses Thereof

Certain aspects of the present invention concern at least one FDHnucleic acid. In certain aspects, the at least one FDH nucleic acidcomprises a wild-type or mutant FDH or nucleic acid. In particularaspects, the FDH or nucleic acid encodes for at least one transcribednucleic acid. In certain aspects, the FDH or nucleic acid comprises atleast one transcribed nucleic acid. in particular aspects, the FDH ornucleic acid encodes at least one FDH or protein, polypeptide orpeptide, or biologically functional equivalent thereof. In otheraspects, the FDH or nucleic acid comprises at least one nucleic acidsegment of the exemplary SEQ ID NO:1, or at least one biologicallyfunctional equivalent thereof.

The present invention also concerns the isolation or creation of atleast one recombinant construct or at least one recombinant host cellthrough the application of recombinant nucleic acid technology known tothose of skill in the art or as described herein. The recombinantconstruct or host cell may comprise at least one FDH or nucleic acid,and may express at least one FDH or protein, peptide or peptide, or atleast one biologically functional equivalent thereof.

As used herein “wild-type” refers to the naturally occurring sequence ofa nucleic acid at a genetic locus in the genome of an organism, andsequences transcribed or translated from such a nucleic acid. Thus, theterm “wild-type” also may refer to the amino acid sequence encoded bythe nucleic acid. As a genetic locus may have more than one sequence oralleles in a population of individuals, the term “wild-type” encompassesall such naturally occurring alleles. As used herein the term“polymorphic” means that variation exists (i.e., two or more allelesexist) at a genetic locus in the individuals of a population. As usedherein “mutant” refers to a change in the sequence of a nucleic acid orits encoded protein, polypeptide or peptide that is the result of thehand of man.

A nucleic acid may be made by any technique known to one of ordinaryskill in the art. Non-limiting examples of synthetic nucleic acid,particularly a synthetic oligonucleotide, include a nucleic acid made byin vitro chemically synthesis using phosphotriester, phosphite orphosphoramidite chemistry and solid phase techniques such as describedin EP 266,032, incorporated herein by reference, or via deoxynucleosideH-phosphonate intermediates as described by Froehler et al., 1986, andU.S. Pat. No. 5,705,629, each incorporated herein by reference. Anon-limiting example of enzymatically produced nucleic acid include oneproduced by enzymes in amplification reactions such as PCR™ (see forexample, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, eachincorporated herein by reference), or the synthesis of oligonucleotidesdescribed in U.S. Pat. No. 5,645,897, incorporated herein by reference.A non-limiting example of a biologically produced nucleic acid includesrecombinant nucleic acid production in living cells, such as recombinantDNA vector production in bacteria (see for example, Sambrook et al.1989, incorporated herein by reference).

A nucleic acid may be purified on polyacrylamide gels, cesium chloridecentrifugation gradients, or by any other means known to one of ordinaryskill in the art (see for example, Sambrook et al. 1989, incorporatedherein by reference).

The term “nucleic acid” will generally refer to at least one molecule orstrand of DNA, RNA or a derivative or mimic thereof, comprising at leastone nucleobase, such as, for example, a naturally occurring purine orpyrimidine base found in DNA (e.g. adenine “A,” guanine “G,” thymine “T”and cytosine “C”) or RNA (e.g. A, G, uracil “U” and C). The term“nucleic acid” encompass the terms “oligonucleotide” and“polynucleotide.” The term “oligonucleotide” refers to at least onemolecule of between about 3 and about 100 nucleobases in length. Theterm “polynucleotide” refers to at least one molecule of greater thanabout 100 nucleobases in length. These definitions generally refer to atleast one single-stranded molecule, but in specific embodiments willalso encompass at least one additional strand that is partially,substantially or fully complementary to the at least one single-strandedmolecule. Thus, a nucleic acid may encompass at least onedouble-stranded molecule or at least one triple-stranded molecule thatcomprises one or more complementary strand(s) or “complement(s)” of aparticular sequence comprising a strand of the molecule. As used herein,a single stranded nucleic acid may be denoted by the prefix “ss”, adouble stranded nucleic acid by the prefix “ds”, and a triple strandednucleic acid by the prefix “ts.”

Thus, the present invention also encompasses at least one nucleic acidthat is complementary to a FDH or nucleic acid. In particularembodiments the invention encompasses at least one nucleic acid ornucleic acid segment complementary to the sequence set forth in, forexample, SEQ ID NO:1. Nucleic acid(s) that are “complementary” or“complement(s)” are those that are capable of base-pairing according tothe standard Watson-Crick, Hoogsteen or reverse Hoogsteen bindingcomplementarity rules. As used herein, the term “complementary” or“complement(s)” also refers to nucleic acid(s) that are substantiallycomplementary, as may be assessed by the same nucleotide comparison setforth above. The term “substantially complementary” refers to a nucleicacid comprising at least one sequence of consecutive nucleobases, orsemiconsecutive nucleobases if one or more nucleobase moieties are notpresent in the molecule, are capable of hybridizing to at least onenucleic acid strand or duplex even if less than all nucleobases do notbase pair with a counterpart nucleobase. In certain embodiments, a“substantially complementary” nucleic acid contains at least onesequence in which about 70%, about 71%, about 72%, about 73%, about 74%,about 75%, about 76%, about 77%, about 77%, about 78%, about 79%, about80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,to about 100%, and any range therein, of the nucleobase sequence iscapable of base-pairing with at least one single or double strandednucleic acid molecule during hybridization. In certain embodiments, theterm “substantially complementary” refers to at least one nucleic acidthat may hybridize to at least one nucleic acid strand or duplex instringent conditions. In certain embodiments, a “partly complementary”nucleic acid comprises at least one sequence that may hybridize in lowstringency conditions to at least one single or double stranded nucleicacid, or contains at least one sequence in which less than about 70% ofthe nucleobase sequence is capable of base-pairing with at least onesingle or double stranded nucleic acid molecule during hybridization.

6. Assays of Gene Expression

Assays may be employed within the scope of the instant invention fordetermination of the relative efficiency of gene expression. Forexample, assays may be used to determine the efficacy of deletionmutants of specific promoter regions in directing expression ofoperatively linked genes. Similarly, one could produce random orsite-specific mutants of promoter regions and assay the efficacy of themutants in the expression of an operatively linked gene. Alternatively,assays could be used to determine the function of a promoter region inenhancing gene expression when used in conjunction with variousdifferent regulatory elements, enhancers, and exogenous genes.

Gene expression may be determined by measuring the production of RNA,protein or both. The gene product (RNA or protein) may be isolatedand/or detected by methods well known in the art. Following detection,one may compare the results seen in a given cell line or individual witha statistically significant reference group of non-transformed controlcells. Alternatively, one may compare production of RNA or proteinproducts in cell lines transformed with the same gene operatively linkedto various mutants of a promoter sequence. In this way, it is possibleto identify regulatory regions within a novel promoter sequence by theireffect on the expression of an operatively linked gene.

In certain embodiments, it will be desirable to use genes whoseexpression is naturally linked to a given promoter or other regulatoryelement. For example, a prostate specific promoter may be operativelylinked to a gene that is normally expressed in prostate tissues.Alternatively, marker genes may be used for assaying promoter activity.Using, for example, a selectable marker gene, one could quantitativelydetermine the resistance conferred upon a tissue culture cell line oranimal cell by a construct comprising the selectable marker geneoperatively linked to the promoter to be assayed. Alternatively, varioustissue culture cell line or animal parts could be exposed to a selectiveagent and the relative resistance provided in these parts quantified,thereby providing an estimate of the tissue specific expression of thepromoter.

Screenable markers constitute another efficient means for quantifyingthe expression of a given gene. Potentially any screenable marker couldbe expressed and the marker gene product quantified, thereby providingan estimate of the efficiency with which the promoter directs expressionof the gene. Quantification can readily be carried out using eithervisual means, or, for example, a photon counting device.

A preferred screenable marker gene for use with the current invention isβ-glucuronidase (GUS). Detection of GUS activity can be performedhistochemically using 5-bromo-4-chloro-3-indolyl glucuronide (X-gluc) asthe substrate for the GUS enzyme, yielding a blue precipitate inside ofcells containing GUS activity. This assay has been described in detail(Jefferson, 1987). The blue coloration can then be visually scored, andestimates of expression efficiency thereby provided. GUS activity alsocan be determined by immunoblot analysis or a fluorometric GUS specificactivity assay (Jefferson, 1987). Similarly, 5-bromo-4chloro-3-indolylgalactoside (X-gal) is often used as a selectable marker, which confersa blue color on those transformants that comprise β-galactosidaseactivity.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those skilled in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1 Methods to Construct Bacterial Strain and Plasmid

The strain BS1 was constructed from the strain GJT001 (Tolentino et al.,1992) by inactivating the native formate dehydrogenase. The exemplaryplasmid pSBF2 contains the fdh1 gene from the yeast Candida boidinii(SEQ ID NO:1) under the control of the lac promoter. The fdh1 geneencodes an NAD+-dependent formate dehydrogenase (FDH) that convertsformate to CO₂ with the regeneration of NADH from NAD+. This is incontrast with the native formate dehydrogenase that converts formate toCO₂ and H₂ with no cofactor involvement (FIG. 2). Also shown in FIG. 2is the conversion of pyruvate to acetyl-CoA and formate by pyruvateformate lyase (PFL) under anaerobic conditions and to acetyl-CoA and CO₂by pyruvate dehydrogenase (PDH) under aerobic conditions.

Recombinant bacterial strains and plasmids used in this study are listedin Table 3.

TABLE 3 Bacterial strains and plasmids Significant genotype StrainsGJT001 Spontaneous cadR mutant of MC4100, Sm^(R) DH10B Cloning host M9sMC4100 φ(fdhF′-′lacZ), Ap^(R) BS1 GJT001 φ(fdhF′-′lacZ), Ap^(R) PlasmidspUC18 Cloning vector, Ap^(R) PDHK29, Control, cloning vector, Km^(R)pDHK30 pFDH1 fdh1 in pBluescriptII SK+ PUCFDH Intermediate plasmid, fdh1in pUC18, Ap^(R) pSBF2 fdh1 in pDHK30, Km^(R)

Strain BS1 was constructed by replacing the wild-type fdhF gene with afdhF′-'lacZ fusion by a P1 vir-mediated phage transduction with E. coliM9s (Pecher et al., 1983) as donor and E. coli GJT001 as recipient. TheP1 phage transduction was performed following standard protocols(Maniatis et al., 1989). Ampicillin resistant transductants wereselected for further analysis. The lack of formate dehydrogenaseactivity was confirmed by a previously described method with minormodifications (Mandrand-Berthelot et al., 1978). Briefly, wild type andtransduced GJT001 were grown on glucose minimal media plates for twodays in an anaerobic chamber under an atmosphere of H₂ and CO₂. Anoverlay solution composed of 0.6% agar, 2 mg/ml benzyl viologen, 0.25Msodium formate and 25 mM KH₂PO₄ (pH 7.0) was poured over the plates. Thepresence of formate dehydrogenase activity in the wild type GJT001 wasevidenced by a change in color of the colonies, which turned purple. Thecolonies of the transductants remained white, thus indicating the lackof formate dehydrogenase activity. The presence of the mutation of fdhFin the transductants was also confirmed by PCR. Primers complementary tothe ends of the fdhF gene (forward primer SEQ ID NO:8,5′-GATTAACTGGAGCGAGACC-3′; reverse primer SEQ ID NO:9,5′-TCCGAAAGGAGGCTGTAG-3′) (Zinoni et al., 1986) were used to amplifythis gene in both wild type and transduced GJT001. The disruption of thefdhF gene in the transduced strain was confirmed by the absence of a PCRproduct as opposed to a 2.2-kb product corresponding to the completegene in the wild type strain.

Plasmid pFDH1 was kindly provided by Dr. Y. Sakai (Sakai et al., 1997).It contains a 3kb EcoRI insert containing the fdh1 gene from the yeastCandida boidinii in pBluescriptII SK+. The fdh1 gene in this plasmid isunder the control of its native promoter. Preliminary experiments withthis plasmid showed no FDH activity, suggesting that fdh1 from the yeastwas not properly expressed in E. coli. For this reason, the open readingframe of the fdh gene from C. boidinii was amplified by PCR and placedunder the control of the lac promoter for overexpression in E. coli.

XL-PCR was performed using the GeneAmp XL PCR kit from PE AppliedBiosystems following the manufacturer's protocol. This kit was chosenbecause of the proofreading ability of the enzyme rTth DNA Polymerase,Polymerase, which not only promotes efficient DNA synthesis but alsocorrects nucleotide misincorporations. Plasmid pFDH1 was used as atemplate and the following were used as forward and reverse primersrespectively: forward primer SEQ ID NO:10, 5′-GCGGAATTCAGGAGGAATTTAAA

AAGATCGTTTTAGTCTTATATGATGCT-3′; reverse primer SEQ ID NO:11, CGCGGATCC

TTTCTTATCGTGTTTACCGTAAGC-3′. An EcoRI and a BamHI site were inserted inthe forward and reverse primers respectively, as represented by theunderlined regions.

The program used for the PCR reaction consisted of an initialdenaturation step at 94° C. for 1 minute and 30 seconds followed by 18cycles of denaturation at 94° C. for 30 seconds and combinedannealing/extension at 55-66° C. for 5 minutes. This was followed by 12cycles in which the annealing/extension time was increased by 15 secondsin each cycle until it reached 8 minutes. A final step at 72° C. for 10minutes concluded the PCR.

The PCR product was verified by agarose gel electrophoresis. It waspurified from the reaction mixture and concentrated following theprotocol of the StrataPrep™ PCR Purification Kit (Stratagene—La Jolla,Calif.). The purified fdh PCR product and the vector pUC18 were digestedwith EcoRI and BamHI. Both fragments were ligated and the ligationproduct was transformed into E. coli strain DH10B. White colonies fromAp/Xgal/IPTG plates were selected for further analysis and miniprepswere performed. Insertion of the fdh gene was confirmed by agarose gelelectrophoresis after digestion with EcoRI/SalI.

This plasmid served as an intermediate vector to facilitate theinsertion of the fdh gene into pDHK30 (Phillips et al., 2000) in theright orientation. It was ultimately desired to have the fdh gene in thepDHK30 backbone because it is a high copy number plasmid with kanamycinresistance, which will not interfere with the ampicillin resistance ofthe BSI strain. An additional advantage of this vector is that it can beco-transformed in a two-plasmid system together with the most commonhigh copy number vectors bearing a ColE1 origin.

The intermediate plasmid containing fdh (pUCFDH), and pDHK30 weredigested with EcoRI/XbaI and ligated to obtain plasmid pSBF2. Theligation product was transformed into DH10B and white colonies fromKm/Xgal plates were analyzed. Minipreps were obtained and analyzed byagarose gel electrophoresis after digestion with EcoRI/XbaI. Anappropriate plasmid was selected and transformed both into GJT001 andthe fdh⁻ strain BS1. Strain GJT001 was also transformed with pDHK29, andBS1 was transformed with pDHK30 to serve as negative controls.

Example 2 FDH Activity Assay

Determining FDH activity of strains GJT001 (pSBF2) and BS1 (pSBF2)comprised growing a culture of cells overnight in LB media supplementedwith 20 g/L glucose and 100 mg/L kanamycin (Km) under anaerobicconditions. The cultures were inoculated with 100 μl of a 5 ml overnightLB culture and grown in a shaker at 37° C. and 250 rpm. Cells wereharvested by centrifugation of 20 ml of culture at 4,000 g and 4° C. for10 minutes. The pellet was suspended in 10 ml of 10-mM sodium phosphatebuffer (refrigerated) at pH 7.5 with 0.1M β-mercaptoethanol andcentrifuged as described above. The cells were resuspended in 10 ml of10-mM sodium phosphate buffer (refrigerated) at pH 7.5 with 0.1Mβ-mercaptoethanol and sonicated for 6 minutes in an ice bath (Sonicator:Heat System Ultrasonics, Inc. Model W-255; Settings: 60% cycle, maxpower=8). The sonicated cells were centrifuged at 1,500 g and 4° C. for60 min to remove cell debris and reduce the NAD background. The formatedehydrogenase activity was assayed at 30° C. by adding 100 μl of cellextract to 1 ml of a reaction mixture containing 1.67 mM NAD+, 167 mMsodium formate and 100 mM β-mercaptoethanol in phosphate buffer pH 7.5and measuring the increase in absorbance of NADH at 340 nm (Schutte etal., 1976) modified). One unit was defined as the amount of enzyme thatproduced 1 μmol of NADH per minute at 30° C. Total protein concentrationin cell extracts was measured by Lowry's method (Sigma Kit) using bovineserum albumin as standard.

Example 3 Growth Experiments: Anaerobic and Aerobic Conditions

Growth experiments were conducted on strains GJT001 (pDHK29) and BS1(pSBF2) by growing aerobically triplicate cultures in a rotary shaker at37° C. and 250 rpm. The cultures were grown in 250-ml shake flaskscontaining 50 ml of LB media supplemented with 10 g/L glucose, 100 mg/Lkanamycin, and 0 or 100 mM formate. The O.D. at 600 nm was measuredevery 30 minutes during the exponential growth phase.

The anaerobic tube experiments were performed using 40-ml or 45-ml glassvials with open top caps and PTFE/silicone rubber septa. Each vial wasfilled with 35 ml (40-ml vials) or 40 ml (45-ml vials) of LB mediasupplemented with 20 g/L glucose, 100 mg/L kanamycin, 0 or 50 mMformate, and 1 g/L NaHCO₃ to reduce the initial lag time that occursunder anaerobic conditions. The triplicate cultures were inoculated with100 μl of a 5 ml LB overnight culture. After inoculation, air (6 ml) wasremoved with a syringe from the headspace to ensure anaerobicconditions. The cultures were grown in a rotary shaker at 37° C. and 250rpm. A sample of the initial media was saved for analysis and sampleswere withdrawn with a syringe at 24 hour intervals (24, 48, and 72 hrs).

The aerobic experiment was performed by growing triplicate culturesaerobically using either 125-ml shake flasks containing 25 ml of LBmedia or 250-ml shake flasks containing 50 ml of LB media. The LB mediawas supplemented with about 10 g/L glucose, 100 mg/L kanamycin, anddifferent amounts of formate. The cultures were inoculated with 50 μl or100 μl of a 5 ml LB overnight culture and grown in a rotary shaker at37° C. and 250 rpm. A sample of the initial media was saved for HPLCanalysis and samples were collected after 24 hours of growth.

Example 4 Methods of Analysis

Cell density (OD) was measured at 600 nm in a Spectronic 1001spectrophotometer (Bausch & Lomb, Rochester, N.Y.). Fermentation sampleswere centrifuged for 5 minutes in a microcentrifuge. The supernatant wasfiltered through a 0.45-micron syringe filter and stored chilled forHPLC analysis. The fermentation products and glucose concentrations werequantified using an HPLC system (Thermo Separation Products, Allschwil,Switzerland) equipped with a cation-exchange column (HPX-87H, BioRadLabs, Hercules, Calif.) and a differential refractive index detector. Amobile phase of 2.5 mM H₂SO₄ solution at a 0.6 ml/min flow rate wasused, and the column was operated at 55° C.

Example 5 FDH Activity

The effect of increasing intracellular NADH availability by geneticengineering on the metabolic patterns of Escherichia coli underanaerobic and aerobic conditions was determined. More specifically, theeffect of regenerating NADH by substituting the nativecofactor-independent formate dehydrogenase in E. coli by theNAD+-dependent FDH from Candida boidinii, as well as the effect ofsupplementing the culture media with formate was demonstrated herein.

Plasmid pSBF2, containing the fdh1 gene from Candida boidinii under thecontrol of the lac promoter, was constructed and characterized bydetermining the activity of the new FDH. Table 4 shows the specific FDHactivity of strains BS1 (pSBF2) and GJT001 (pSBF2) in Units/mg of totalprotein. One unit is defined as the amount of enzyme that produced 1μmol of NADH per minute at 30° C. Values shown are average oftriplicates from anaerobic tube cultures. N.D.: not detected (less than0.001 U/mg). The FDH activity of strain GJT001 (pSBF2) was 46% higher(0.416 U/mg) than the activity of BS1 (pSBF2) (0.284 U/mg). Controlstrains GJT001 (pDHK29) and BS1 (pDHK30) showed no detectable FDHactivity.

TABLE 4 Specific FDH activity Strain Activity (U/mg) BS1 (pSBF2) 0.284 ±0.002 GJT001 (pSBF2) 0.416 ± 0.004 GJT001 (pDHK29) N.D. BS1 (pDHK30)N.D.

The effect of substituting the native FDH with the NAD+-dependentpathway was characterized by calculating the specific growth rate (μ) ofstrains BS1 (pSBF2) and GJT001 (pDHK29) in aerobic shake flaskexperiments. Table 5 presents the results of these experiments with andwithout 100 mM formate. The specific growth rate of strain BS1 (pSBF2)was 35% lower (0.986±0.002) than that of GJT001 (pDHK29) (1.511±0.016)without formate supplementation. However, by the end of the fermentationthe cell density of BS1 (pSBF2) was comparable to or even higher thanthat of GJT001 (pDHK29).

In addition, the effect on the specific growth rate of formatesupplementation at the level of 100 mM was examined. Formate addition tothe media lengthened the duration of the lag phase for both strains, butmore for BS1 (pSBF2). The difference in the specific growth rate betweenBS1 (pSBF2) and GJT001 (pDHK29) decreases with addition of formate.Under these conditions, the specific growth rate of GJT001 (pDHK29) isonly 10% higher. Addition of formate did not affect significantly thespecific growth rate of BS1 (pSBF2), however; it decreased that ofGJT001 (pDHK29) by 28%. As in the case without formate supplementation,the final cell density of BS1 (pSBF2) was comparable to that of GJT001(pDHK29).

TABLE 5 Specific aerobic growth rate (μ) of strains BS1 (pSBF2) andGJT001 (pDHK29). Strains: 0 mM Formate 100 mM Formate BS1(pSBF2) 0.986 ±0.002 0.972 ± 0.014 GJT001(pDHK29) 1.511 ± 0.016 1.086 ± 0.043 Valuesshown are average of triplicates.

Example 6 Increased Intracellular NADH Availability and AlcoholProduction During Anaerobiosis

Anaerobic tube experiments were performed with strains GJT001 (pDHK29),GJT001 (pSBF2), BS1 (pSBF2), and BS1 (pDHK30) to investigate the effecton the metabolic patterns of the elimination of the native FDH and theaddition or substitution of the new FDH. FIG. 3A to 3F illustrate theresults of these experiments, including the final cell density (FIG.3A), the amount of glucose consumed in millimolar (mM) (FIG. 3B), andthe concentrations of different metabolites produced (mM) after 72 hoursof culture (FIG. 3C to 3F). Values shown are the average of triplicatecultures.

A comparison of the results for the control strains GJT001 (pDHK29) andBS1 (pDHK30) shows the effect of eliminating the native FDH on themetabolic patterns of E. coli. An increase in residual formate wasobserved for the strain lacking FDH activity. As shown in FIG. 3B,glucose consumption for BS1 (pDHK30) decreased by 47% relative to GJT001(pDHK29). This led to a decrease in final cell density (29%; FIG. 3A),as well as, in succinate (39%; FIG. 3E), lactate (66%; FIG. 3F), andethanol (22%; FIG. 3C) production. However, the level of acetate (FIG.3D) was very similar to that of GJT001 (pDHK29). This translates into adecrease (24%) in the ethanol to acetate (Et/Ac) ratio. This decrease inthe Et/Ac ratio together with the decrease in other reduced metabolites(lactate and succinate) indicates the presence of a more oxidizedenvironment for the strain lacking formate dehydrogenase activity. Theseresults suggest that under normal conditions GJT001 (pDHK29) recapturesa portion of the H₂ produced from the degradation of formate by thenative FDH possibly by means of some hydrogenase, and this recaptureaccounts for the slightly more reduced intracellular environmentobserved for this strain relative to BS1 (pDHK30).

Table 6 gives the quantitative amounts of NADH in terms of(NADH)_(U)/G1=moles of NADH available for reduced product formation permole of glucose consumed, where (NADH)_(U)=Total NADH used for productformation per unit volume at the end of fermentation (mmol/L) and wasestimated from the concentrations of reduced metabolites by calculatingthe NADH used for their production according to the pathways shown onFIG. 1, with 50 mM initial formate supplementation. Values shown arefrom average of triplicate cultures.

TABLE 6 NADH availability of various strains under anaerobic conditions.(NADH)_(U)/Gl Strain (mol/mol) GJT001 (pDHK29) 2.40 GJT001 (pSBF2) 4.34BS1 (pSBF2) 4.35 BS1 (pDHK30) 2.38 GJT001 (pDHK29) + F 2.33 BS1(pSBF2) + F 4.39

An analysis of the results for BS1 (pSBF2) relative to BS1 (pDHK30) andfor GJT001 (pSBF2) relative to GJT001 (pDHK29) provides an understandingof the effect of overexpressing the NAD+-dependent FDH both alone or inconjunction with the native FDH, respectively. In both cases the trendis similar, but the effect is more pronounced for the BS1 strains due tothe decrease in the metabolites observed for BS1 (pDHK30) relative toGJT001 (pDHK29). Both strains containing the new FDH present asignificant increase in glucose consumption, cell density, ethanol, andsuccinate formation, accompanied by a decrease in lactate and acetaterelative to the control strains. This translates into a dramaticincrease in the ethanol to acetate (Et/Ac) ratio of 22-fold for GJT001(pSBF2) and 35 to 36-fold for BS1 (pSBF2).

The results for GJT001 (pSBF2) and BS1 (pSBF2) show the effect of havingboth the native and new FDH active in the same strain or just the newFDH, respectively. A comparison of these results shows that thesestrains behave very similarly. The largest difference between these twostrains is a 16% decrease in acetate, and consequently a 21% increase inEt/Ac ratio for BS1 (pSBF2) relative to GJT001 (pSBF2). This means thatthe NAD+-dependent FDH is competing effectively with the native FDH foravailable formate. This finding is supported by the fact that the Kmvalue for formate of the native FDH is twice (26 mM) that of theNAD+-dependent FDH (13 mM) according to the literature (Schutte et al.,1976; Axley and Grahame, 1991). Although these results suggest that thefdh⁻ mutation is not necessary to observe the effect of overexpressingthe NAD+-dependent FDH, the decrease in acetate levels observed for thefdh⁻ strain suggests that this mutation is be slightly beneficial insome cases.

Analyzing the results of BS1 (pSBF2) relative to GJT001 (pDHK29) canbetter elucidate the effect of substituting the cofactor-independentnative formate-degradation pathway in E. coli by the NAD+-dependentpathway. Substitution of the native FDH by the new FDH increased glucoseconsumption (3-fold), final cell density (59%), as well as theproduction of ethanol (15-fold) and succinate (55%), while it decreasedlactate (91%) and acetate (43%) production (see FIG. 3C to 3F). Thistranslates into a dramatic increase in the ethanol to acetate (Et/Ac)ratio of 27-fold (FIG. 4B).

These results suggest that overexpression of the NAD+-dependent FDHincreases intracellular NADH availability, and this in turn leads to adrastic shift in the metabolic patterns of E. coli. An increase in NADHavailability favored the production of more reduced metabolites,particularly, those requiring 2NADH molecules per molecule of productformed, like ethanol and succinate. The preferred product was ethanol,with a final concentration reaching as high as 175 mM for BS1 (pSBF2),as compared to 11.5 mM for the wild type control, GJT001 (pDHK29). Thismakes ethanol the major fermentation product for BS1 (pSBF2) anaerobiccultures, accounting for 91% of the metabolites produced based on mMconcentrations, as opposed to 18% for GJT001 (pDHK29). Simultaneously,lactate was converted from a major product, representing 57% of theproduced metabolites in the wild type strain to only a minor product,accounting for less than 2% of the metabolites. This shift towards theproduction of ethanol as a major product is comparable to that obtainedwith overexpression of the ethanologenic enzymes from Zymomonas mobilisin the pet operon in E. coli (Ingram and Conway, 1988). Remarkably,these results indicate a significant production of ethanol despite thelack of overexpression of enzymes specifically directed towards ethanolproduction.

The dramatic increase in ethanol production combined with a decrease inacetate levels led to the drastic increase in the Et/Ac ratio observed,which reached as high as 27 for BS1 (pSBF2), as compared to 1.0 forGJT001 (pDHK29). It is evident from these results that the cell adjustsits partitioning at the acetyl-CoA node by changing the ethanol(consumes 2 NADH) to acetate (consumes no NADH) ratio to achieve a redoxbalance, as was previously observed in experiments utilizing carbonsources with different oxidation states (San et al., 2001). Thesefindings also support the idea that NADH induces expression of alcoholdehydrogenase (adhE) (Leonardo et al., 1996).

The significant decrease in lactate levels obtained with overexpressionof the NAD+-dependent FDH can be explained by noting that althoughlactate formation also requires NADH, it only consumes 1 NADH, whileethanol formation consumes 2 NADH. These results suggest that when thereis an excess of reducing equivalents, ethanol formation (2 NADH) ispreferred over lactate formation (1 NADH) since it provides a fasterroute to NAD+ regeneration. These observations support previous findingsin experiments utilizing carbon sources with different oxidation states(San et al., 2001).

FIG. 3A to 3F and FIGS. 4A and 4B illustrate the results of anaerobictube experiments performed with strains GJT001 (pDHK29) and BS1 (pSBF2)in which the media was supplemented with 50 mM formate. Addition offormate to both strains increased lactate levels. A comparison of theresults for BS1 (pSBF2) and GJT001 (pDHK29) indicates a 6-fold increasein ethanol accompanied by a 69% decrease in acetate levels. This leadsto a 21-fold increase in the Et/Ac ratio with the substitution of thenative FDH for the NAD+-dependent FDH. This means that anacrobically itis not necessary to supplement the culture with formate.

The amounts of formate converted to CO₂ for the different strains, withand without formate addition under anaerobic conditions, were calculatedby subtracting the measured residual formate concentration from theconcentration of formate produced plus the initial formate concentrationin the media for the experiments with formate supplementation. Theamount of formate produced was obtained based on the assumption that onemol of formate is produced per mol of acetyl-CoA formed through the PFLpathway (see FIG. 2). Therefore, the amount of formate produced wascalculated by adding the concentrations of ethanol and acetate formedfrom acetyl-CoA.

The data indicate that overexpression of the NAD+-dependent FDHdrastically increases the conversion of formate almost equally for bothstrains BS1 (pSBF2) and GJT001 (pSBF2) suggesting that this new enzymecompetes very effectively with the native FDH for the available formate.These two strains as well as GJT001 (pDHK29) converted all the formateproduced during fermentation when there was no external formate added tothe media, while strain BS1 (pDHK30) converted only minimal amounts offormate as expected.

It is also interesting to note that external addition of formate to themedia had opposite effects on the native and new FDH. Formatesupplementation of GJT001 (pDHK29) cultures significantly increased (2to 3-fold) the amount of formate converted by the native enzyme,although only 78% of the available formate was converted. These resultssuggest that addition of extra formate has a stimulatory effect on thispathway or that initially the pathway was limited by the amount offormate, while after formate supplementation it became limited by theenzyme activity instead. In contrast, addition of formate to BS1 (pSBF2)anaerobic cultures decreased the amount of formate converted, with only69% of the available formate being degraded, suggesting possibleinhibition of the new FDH at these levels of formate. Plausibly, thisdecrease in formate conversion is the indirect consequence of a lowerglucose consumption and optical density. Although the total levels offormate produced for this strain without external formate addition werehigher than with the 50 mM supplementation, the cells did not experiencehigh levels of formate at a given time because it is being degraded asit is produced. In contrast, in the supplementation experiment, the cellexperienced a higher initial formate concentration.

Example 7 Increased Intracellular NADH Availability During Aerobiosis

Shake flask experiments were performed with strains GJT001 (pDHK29) andBS1 (pSBF2) to investigate the effect of increasing intracellular NADHavailability by substituting the native FDH in E. coli by theNAD+-dependent enzyme on the metabolic patterns under aerobicconditions. These experiments were performed with and without 100 mMformate supplementation. Addition of formate as a substrate for the newFDH during aerobic growth was necessary because under these conditionsthe cells normally do not produce formate due to lack of activity of thepyruvate formate lyase (PFL) enzyme. FIG. 5A to 5F presents the resultsof these experiments, including the final cell density (FIG. 5A),glucose consumed (mM) (FIG. 5B), and the concentrations of differentmetabolites produced (mM) after 24 hours of culture (FIG. 5C to 5F). Forboth strains only minimal amounts of residual formate (less than 6 mM)were detected.

This data indicate that addition of formate to BS1 (pSBF2) aerobiccultures induced the production of ethanol, lactate, and succinate,metabolites that are normally produced only under anaerobic conditions.The amount detected corresponds to a 36-fold increase in ethanol (FIG.5C), 7-fold increase in succinate (FIG. 5E), and the production oflactate (FIG. 6A). Glucose consumption increased by 50% and acetatelevels by 11%. The Et/Ac ratio increased by 32-fold.

Also addressed by this data is the effect of formate supplementation onthe native FDH was also investigated. Addition of formate to GJT001(pDHK29) aerobic cultures caused an increase of 50% in glucoseconsumption, the same percentage of increase observed for BS1 (pSBF2).However, the increase in acetate levels was much higher (47%) withformate supplementation, as well as the increase in final cell density(48%). On the other hand, the production of ethanol was much lower, only5.15 mM after 24 hours, and succinate levels increased only by 72%compared to a 7-fold increase for BS1 (pSBF2).

The results obtained for both strains with formate supplementation showsa 27-fold increase in lactate, 4-fold increase in ethanol, 3-foldincrease in succinate, accompanied by a 30% decrease in acetate (5-foldincrease in Et/Ac) for the NAD+-dependent FDH relative to the nativeFDH. The glucose consumption was similar for both strains, while thefinal cell density was slightly higher for BS1 (pSBF2).

These results demonstrate that it is possible to increase theavailability of intracellular NADH through the substitution of thenative FDH in E. coli by an NAD+-dependent FDH. The higher intracellularNADH levels provide a more reduced environment even under aerobicconditions. As a result, the cells utilize this extra NADH to reducemetabolic intermediates leading to the formation of fermentationproducts in order to achieve a redox balance. Conversely, under normalaerobic conditions, the environment is so oxidized that reducedfermentation products are not formed. Under aerobic conditions, onlyacetate, a more oxidized metabolite that does not require NADH, isnormally produced. The results described herein also suggest thatalthough the native FDH is able to indirectly recapture some of theextra reducing power in the formate added, the new FDH is a lot moreeffective because it recaptures this extra reducing power directly asNADH.

Example 8 Effect of Formate on Reduction Processes During Aerobiosis

In addition, the effect of supplementing the media with different levelsof formate (0, 50, 100, 150, and 200 mM) was investigated in aerobiccultures of BS1 (pSBF2). It is interesting to note that lactate wasabsent at 0 and 50 mM initial formate, but it was produced at 100, 150,and 200 mM initial formate (FIGS. 6A and 6B). The concentration oflactate increased with an increase in the initial formate levels. Thesame trend was evident in succinate production with the difference thatsimilar levels were produced at 0 and 50 mM initial formate (FIG. 6B).On the other hand, the cells produced ethanol only after formatesupplementation, but the levels did not significantly increase with anincrease in formate levels. Acetate production and final cell densitydid not follow any notable trend with increasing levels of formatesupplementation. Glucose consumption increased with addition of formateand remained constant with different formate levels because all theglucose was consumed by 24 hours in all the formate supplementedcultures.

It was also observed that the concentration of residual formate reached63.5 mM for the 200 mM initial formate experiment, a 10-fold increasefrom the residual levels in the 150 mM experiment. The levels ofresidual formate were lower than 12 mM for all other initial formatelevels. These findings possibly indicate that the culture is pastsaturation at this formate level. In addition, based on the formateconversion levels observed, more NADH is being generated by this pathwaythan that used to produce reduced metabolites. The cells are possiblyusing this extra NADH formed for ATP generation through the electrontransport system since they are growing aerobically.

The results of the formate supplementation experiment show thatdifferent formate levels can be used to provide different levels ofreducing power. Higher levels of reducing power aerobically mainlyincreased lactate production. In contrast, in anaerobic cultures with noformate supplementation, where the environment was a lot more reduced,ethanol production was highly increased, while lactate levels decreased.However, formate supplementation in anaerobic cultures provoked anincrease in lactate levels, which is consistent with the aerobic case.

Example 9 Increasing Reductive Capabilities In Vivo

The data indicates that it is possible to increase the availability ofintracellular NADH through metabolic engineering, thereby providingenhanced reducing power under both anaerobic and aerobic conditions.

The substitution of the native cofactor independent FDH pathway by theNAD+-dependent FDH provoked a significant metabolic redistribution bothanaerobically and aerobically. Under anaerobic conditions, the increasedNADH availability favored the production of more reduced metabolites, asevidenced by a dramatic increase in the ethanol to acetate ratio for BS1(pSBF2) as compared to the GJT1 (pDHK29) control (FIG. 4B). This led toa shift towards the production of ethanol as the major fermentationproduct (FIG. 3C).

Further during aerobic growth, the increased availability of NADHinduced a shift to fermentation even in the presence of oxygen bystimulating pathways that are normally inactive under these conditions.Because formate is not a normal product under aerobic conditions, it wasadded to the media to increase NADH availability. The addition offormate to BS1 (pSBF2) aerobic cultures induced the production ofethanol, lactate, and succinate, metabolites that are normally producedonly under anaerobic conditions.

Example 10 Chemostat Cultures

The novel approach to increasing availability of intracellular NADH invivo through a NADH recycling system is applied to the production ofcommercially viable compounds, such as ethanol. The NADH recyclingsystem comprises a biologically active NAD+-dependent formatedehydrogenase (FDH) from Candida boidinii, and overexpression thereof inEscherichia coli. The NADH recycling system (e.g., recombinant formatedehydrogenase pathway) produces one mole of NADH per one mole of formateconverted to carbon dioxide (FIG. 2). This recombinant system bearscontrast with the native formate dehydrogenase which converts formate toCO₂ and H₂ with no cofactor involvement. The new NADH recycling systemallows the cells to retain the reducing power that are otherwise lost byrelease of formate or hydrogen.

The functionality of this approach was further characterized byevaluating anaerobic chemostat cultures in a controlled bioreactorenvironment.

Example 11 Methods of Anaerobic Chemostat Experiments

Initially, the inoculum was grown as a 5-ml LB culture supplemented with100 mg/L ampicillin and/or kanamycin for 8-12 hours. Then, 100 μl of the5-ml culture was transferred to 50 ml of LB in a 250-ml shake flask withthe appropriate antibiotic, and grown at 37° C. and 250 rpm for 8-12hours in a rotary shaker. This culture was used to inoculate thebioreactor.

Luria-Bertani broth (LB) medium supplemented with 110 mM of glucose, wasused for the chemostat runs. To reduce the initial lag time that occursunder anaerobic conditions, 1 g/L NaHCO₃ was added to the LB media. Themedia was also supplemented with 30 μL/L antifoam 289 (Sigma), 100 mg/Lampicillin, and/or kanamycin.

The fermentations were carried under anaerobic chemostat conditions at adilution rate of 0.2 hr⁻¹. A 2.5 L bioreactor (New Brunswick Scientific,Bioflo III) was used. It initially contained 1.3 L of medium during theanaerobic batch stage and then was maintained at 1.20 L working volumefor the anaerobic chemostat stage. The pH, temperature and agitationwere maintained at 7.0, 32° C., and 250 rpm, respectively. A constantflow of nitrogen (10-12 ml/min) was maintained through the fermentorheadspace to establish anaerobic conditions. The continuous culturereached steady state after 4 to 6 residence times. Samples were takenduring the steady state phase.

Cell dry weight was determined by collection of 100 ml of culture in anice bath. The samples were centrifuged at 4,000 g and 4° C. for 10minutes, washed with 0.15M sodium chloride solution, and dried in anoven at 55° C. until constant weight. The final weight of the driedsamples was corrected for the weight of NaCl in the washing solution.

For chromatography, samples of the fermentation broth were collected andcentrifuged at 6000 g and 4° C. for 10 minutes in a Sorvall centrifuge(SS-34 rotor).

Example 12 FDH Activity in Anaerobic Chemostat Cultures

Experiments were performed under anaerobic chemostat conditions withstrains GJT001 and BS1 containing a control plasmid to investigate theeffect of eliminating the native formate dehydrogenase activity. Theresults of those experiments indicated that with inactivation of thenative FDH, which converts formate to CO₂ and H₂, reducing power is lostin the form of formate. This resulted in a more oxidized intracellularenvironment as reflected by a significant decrease in the NADH/NAD+ratio (48%) and a decrease in the Et/Ac ratio (19%). These observationsarc consistent with previously reported results under anaerobic tubeconditions with these two strains. These results imply that under normalconditions when the native FDH is active, the cells are able torecapture some of the reducing power in the hydrogen released from thedegradation of formate possibly by means of a native hydrogenase. Thesefindings suggest that substitution of the native FDH by anNAD+-dependent FDH, which transfers the reducing equivalents directlyfrom formate to NADH, provides a more reduced intracellular environmentby recapturing more effectively the reducing power that otherwise islost.

Anaerobic chemostat experiments were performed with strains GJT001(pSBF2), BS1 (pSBF2), and GJT001 (pDHK29). Strain GJT001 (pDHK29)contains the native formate dehydrogenase (FDH) only, while strain BS1(pSBF2) has the C. boidinii FDH, and GJT001 (pSBF2) has both FDH enzymesactive. A chemostat mode was chosen because it allows the determinationof the concentration of NADH and NAD+ and the metabolic fluxes duringsteady state. It also allows fixing of the specific growth rate for eachstrain by fixing the dilution rate (0.2 h⁻¹).

Table 7 presents the specific NAD+-dependent FDH activity of strainsGJT001 (pSBF2) and BS1 (pSBF2) obtained from the anaerobic chemostatruns in units/mg of total protein. One unit is defined as the amount ofenzyme that produced 1 μmol of NADH per minute at 30° C. As this tableshows, the specific FDH activity of both strains was very similar.Strain GJT001 (pDHK29) showed no detectable FDH activity. One unit isdefined as the amount of enzyme that produced 1 μmol of NADH per minuteat 30° C. Values shown are average of triplicates. N.D.: not detected(less than 0.001 U/mg).

TABLE 7 Specific NAD+-dependent FDH activity. Strain Activity (U/mgTP)GJT001 (pSBF2) 0.242 ± 0.009 BS1 (pSBF2) 0.231 ± 0.007 GJT001 (pDHK29)N.D.

Example 13 Metabolic Flux Redistribution in Anaerobic Chemostat Cultures

Steady state concentrations of metabolites are given in Table 8 asmillimolar (mM) units as measured by the HPLC, as well as the percent ofCO₂ and H₂ per volume in the off-gases stream as measured by the GC. Theconcentrations are in anaerobic chemostat (average of three samples) atD=0.2 h⁻¹. CO₂ and H₂ in % per volume as measured from the off-gases byGC. Dry weight (D.W.) in g/L.

Table 9 presents the results as calculated metabolic fluxes in mmol/(gdry weight*h) represented as v₁ to v₁₂ according to the diagramillustrated in FIG. 8. Note that v₁₂ represents the newly addedNAD+-dependent FDH pathway. In addition, v_(RF) represents the flux ofresidual formate excreted to the media based on HPLC measurements. Themetabolic fluxes with an asterisk were calculated based on measuredmetabolites, while the other fluxes were derived from the measuredmetabolites based on the relationships shown in FIG. 8, the law of massconservation, and the pseudo-steady-state hypothesis (PSSH) on theintracellular intermediate metabolites as described previously(Aristidou et al., 1999; Yang et al., 1999). Metabolic fluxes with anasterisk were calculated based on measured metabolites, while the otherfluxes were derived from the measured metabolites based on therelationships shown in FIG. 8. The percentages of increase (+) ordecrease (−) presented are relative to strain GJT001 (pDHK.29). The “+”indicates that the culture comprised the newly added NAD+-dependent FDHpathway.

Table 10 includes the NAD(H/+) concentrations in μmol/g dry weight(D.W.) in addition to the NADH formed through the oxidation of glucoseand the new FDH degradation pathway, as well as the NADH utilized forthe formation of reduced metabolites, namely, succinate, lactate, andethanol. The percentages of increase (+) or decrease (−) presented onthese tables are relative to strain GJT001 (pDHK29), and are an averageof three samples at a dilution, D=0.2 h⁻¹. (R_(NADH))_(f)=specific NADHformation rate=v₄+v₁₂; (R_(NADH))_(u)=specific NADH utilizationrate=2v₆+v₇+2v₁₀. Both rates are in units of mmol/(gD.W.*h). Thepercentages of increase (+) or decrease (−) are determined relative tostrain GJT001 (pDHK29).

The overexpression of the NAD+-dependent FDH drastically changed thedistribution of metabolic fluxes in E. coli. The most notable effectobserved is the shift in the ethanol to acetate ratio (Et/Ac), whichindicates an increase in intracellular NADH availability. This ratioincreased from 1.06 for the control strain to 3.47 for the strain withthe new FDH and 3.82 for the strain with both enzymes coexpressed. Thisrepresents a 3 to 4-fold increase in the Et/Ac ratio relative to thecontrol. These findings are similar to the results obtained whensorbitol (Et/Ac=3.62), a more reduced carbon source that can thereforeproduce more reducing equivalents in the form of NADH, was used insteadof glucose (Et/Ac=1.00) in anaerobic chemostat experiments (San et al.,2001).

TABLE 8 Metabolite concentrations of recombinant strains. Strain GJT001GJT001 BS1 (pDHK29) (pSBF2) (pSBF2) Glucose 113.36 ± 0.59  94.74 ± 3.99 64.43 ± 4.98 Consumed Succinate 13.50 ± 0.31 9.49 ± 1.33  5.05 ± 0.46Lactate 37.37 ± 0.60 4.38 ± 0.41  1.96 ± 0.32 Residual 64.35 ± 0.9636.89 ± 3.66  43.91 ± 2.04 Formate Acetate 74.26 ± 0.77 35.75 ± 2.74 25.88 ± 1.09 Ethanol 78.86 ± 0.97 136.54 ± 8.78  89.70 ± 6.62 Et/Ac 1.063.82 3.46 CO₂ 11.58 ± 0.38 16.25 ± 2.89  10.71 ± 0.93 H₂ 16.95 ± 0.057.01 ± 0.59  0.02 ± 0.02 D.W.  2.48 ± 0.03 1.31 ± 0.01  2.03 ± 0.08

TABLE 9 Anaerobic chemostat results. GJT001 GJT001 % Inc/ BS1 % Inc/Flux To: (pDHK29) (pSBF2) Dec (pSBF2) Dec ν₁ Glucose Uptake* 7.81 12.9365.63 5.53 −29.20 ν₂ Biosynthesis 0.78 0.23 −71.06 0.27 −65.61 ν₃Glyceraldehyde 3-P 7.03 12.71 80.87 5.26 −25.14 ν₄ PEP 14.05 25.42 80.8710.52 −25.14 ν₅ Pyruvate 13.12 24.12 83.81 10.09 −23.13 ν₆ Succinate*0.93 1.30 39.38 0.43 −53.41 ν₇ Lactate* 2.57 0.60 −76.76 0.17 −93.48 ν₈Formate 10.55 23.52 123.00 9.92 −5.97 ν₉ H₂* 6.12 1.55 −74.70 0.00−99.95 ν_(RF) Residual Formate* 4.43 5.04 13.63 3.77 −15.00 ν₁₀ Ethanol*5.43 18.64 243.15 7.70 41.71 ν₁₁ Acetate* 5.12 4.88 −4.59 2.22 −56.59ν₁₂ New FDH Pathway⁺ 0.00 16.94 — 6.15 —

TABLE 10 Anaerobic chemostat results. Strain GJT001 GJT001 % Inc/ BS1 %Inc/ (pDHK29) (pSBF2) Dec (pSBF2) Dec NADH 6.64 6.40 −3.60 5.53 −16.70NAD+ 6.27 5.90 −5.95 4.34 −30.74 NADH/NAD+ 1.06 1.09 2.74 1.29 21.42Total NAD(H/+) 12.90 12.29 −4.74 9.87 −23.52 (R_(NADH))_(f) 14.05 42.35— 16.67 — (R_(NADH))_(u) 15.30 40.47 — 16.43 — (NADH)_(U)/Gl 1.96 3.1359.69 2.97 51.53

Example 14 NADH/NAD+ Ratio

Importantly, the effect of the cofactor manipulations is smaller underchemostat conditions as compared to previous findings in anaerobic tubeexperiments (Et/Ac=27.0 for BS1 (pSBF2)). This is explained by thedifference in the growth environment and conditions the cells areexposed to in a batch versus chemostat cultivation. In a chemostatbioreactor the specific growth rate equals the dilution rate, is fixedexternally and is dependent on the strain and media composition for abatch culture. In addition, the transient nature of the batchcultivation implies that the concentration of both substrates andmetabolites varies constantly with time, while at steady state theseconcentrations are time-invariant for a chemostat culture. Specifically,the cells are exposed to a very rich environment for most of the timeduring batch cultivation, while they are always under limitingenvironment under a chemostat setting. A similar behavior was observedpreviously in experiments where a significant acetate reduction wasachieved under batch conditions by modulating glucose uptake using aglucose analog supplementation strategy, however the effect was greatlyminimized under chemostat conditions (Chou et al., 1994).

The current results support previous findings (San et al., 2001) thatthe cell adjusts its partitioning at the acetyl-CoA node by changing theethanol (consumes 2 NADH) to acetate (consumes no NADH) ratio to achievea redox balance. Therefore, a change in the ethanol to acetate ratio(Et/Ac) is used as an indirect indicator of a change in the NADH/NAD+ratio.

In the chemostat experiments, the NADH/NAD+ ratio increased slightly instrain BS1 (pSBF2), and it remained relatively unchanged for GJT001(pSBF2) as compared to GJT001 (pDHK29). These results suggest that thecells regenerate the extra reducing power in the form of NADH that wasavailable from the overexpression of the new FDH by increasing the fluxto ethanol, which consumes 2 NADH, instead of accumulating the NADH assuch. These findings might indicate that the NADH/NAD+ ratio is notalways a good indicator of the oxidation state of the cell because in aneffort to achieve a redox balance, the turnover is fast. This idea issupported by the fact that more than 96% of the NADH formed through theoxidation of glucose and the new FDH degradation pathway,(R_(NADH))_(f,) can be accounted for as being utilized for the formationof reduced metabolites, namely, succinate, lactate, and ethanol,(R_(NADH))_(u) (Table 10). In addition, the specific NADH formation andutilization rates for both strains containing the new FDH aresignificantly higher than those of the control strain (Table 10).

Example 15 Effect of Redistributing Metabolic Flux

An analysis of the metabolic fluxes of the two experimental strainsrelative to the control strain shows a significant increase in the fluxto ethanol, accompanied by a decrease in the flux to acetate and amarked decrease in the flux to lactate. The increase in the ethanol flux(2 NADH) in combination with the decrease in the flux to lactate (1NADH) indicate that when there is an excess of reducing equivalents,ethanol formation is preferred since it provides a faster route to NAD+regeneration. These results are in agreement with our previous findingsin chemostat experiments utilizing carbon sources with differentoxidation state (San et al., 2001). In those experiments, the lactateflux was highest for gluconate, a more oxidized carbon source, andlowest for sorbitol, a more reduced carbon source relative to glucose.

Tn addition, Table 9 presents the flux of formate converted to CO₂through both the native FDH pathway (v₉) and the new NAD+-dependent FDHpathway (v₁₂) for the different strains. The flux to formate wasobtained based on the assumption that one mole of formate is producedper mole of acetyl-CoA formed through the PFL pathway (FIG. 9).Therefore, the flux to formate (v₈) was calculated by adding the fluxesto ethanol (v₁₀) and acetate (v₁₁) from acetyl-CoA. The total formateconverted was calculated by subtracting the measured residual formateflux (v_(RF)) from the flux to formate (v₈). The flux through the newFDH pathway (v₁₂) for strain GJT001 (pSBF2) was determined bysubtracting the flux to H₂ (v₉), determined from GC measurements, fromthe total formate converted.

The absence of H₂ production as determined by GC analysis of theoff-gases (Tables 7 and 8) confirmed the lack of native formatedehydrogenase activity in strain BS1 (pSBF2). For strain GJT001 (pSBF2),in which both FDH enzymes are active, 92% of the total formate convertedto CO₂ was degraded through the NAD+-dependent FDH pathway. This resultindicates that the new FDH enzyme competes very effectively with thenative FDH for the available formate. This finding is consistent withthe reported Km value for formate of the native FDH being twice (26 mM)that of the NAD+-dependent FDH (13 mM) (Schutte et al., 1976; Axley andGrahame, 1991).

Coexpression of both FDH enzymes in strain GJT001 (pSBF2) increasedglucose uptake under chemostat conditions relative to the controlstrain. However, a decrease in glucose uptake was observed under thesame conditions for strain BS1 (pSBF2). Due to the difference observedin glucose uptake, the yields in carbon-mole produced per carbon-mole ofglucose consumed were calculated for the different metabolites. Thisallows a better understanding of how one carbon-mole (C-mole) of glucoseconsumed by the cell is distributed to the production of the differentmetabolites in each of the strains studied.

Example 16 Effect on Fermentation Products

The calculated yields for the different fermentation products are givenin C-mole produced per C-mole of glucose consumed on FIG. 9. Valuesshown are yields in C-mole produced per C-mole of glucose consumed. Thestrains are identified as follows: GC=GJT001 (pDHK29), GF=GJT001(pSBF2), and BF=BS1 (pSBF2). Results were obtained from anaerobicchemostat experiments at a dilution rate of 0.2 hr⁻¹. Unexpectedly, thepercentage of carbon recovery obtained without accounting for thebiomass was 90% or higher for all the strains.

For the control strain GJT001 (pDHK29), one C-mole of glucose isdistributed almost equally to ethanol (0.23), acetate (0.22), andformate (0.23). The rest of it goes mostly to lactate (0.16), withsuccinate (0.06) being only a minor product. In contrast, for thestrains containing the new FDH pathway, almost half of each C-mole ofglucose was directed towards ethanol production (GJT001 (pSBF2): 0.48,BS1 (pSBF2): 0.46), while the yield to acetate decreased to 0.13, andthat of formate increased (0.30) for both strains. At the same time,lactate proportion decreased to that of a minor product with a yield aslow as 0.02. This yield is even lower than the yield of succinate, whichremained relatively unchanged. It is important to note that thedistribution of C-mole yields for strains GJT001 (pSBF2) and BS1 (pSBF2)is almost identical. This finding implies that under the experimentalconditions studied the native FDH does not interfere with the action ofthe new FDH of redistributing the metabolic fluxes on a C-mole basis.

FIG. 9 also shows the amount of formate produced that is convertedthrough either one or both of the FDH pathways. For the control strain,57% of the formate produced is converted, while 80% is converted forGJT001 (pSBF2) and 63% for BS1 (pSBF2). These results show an increasein the conversion of formate with the overexpression of the new FDH,further suggesting that the new FDH has higher activity or higheraffinity for formate than the native cofactor independent FDH.

Example 17 Recombinant FDH Competes with Native FDH

The reductive capabilities of the chemostat cultures further demonstratean increase in the availability of intracellular NADH through metabolicengineering and therefore provide a more reduced environment underanaerobic chemostat conditions. The substitution of the native cofactorindependent FDH pathway by the NAD+-dependent FDH provoked a significantredistribution of both metabolic fluxes and C-mole yields underanaerobic chemostat conditions.

The increased NADH availability favored the production of more reducedmetabolites, as evidenced by a 3 to 4-fold increase in the ethanol toacetate ratio for BS1 (pSBF2) and GJT001 (pSBF2) as compared to the GJT1(pDHK29) control. This was the result of an increase in the ethanolyield combined with a decrease in the acetate yield. It was alsoobserved that the flux to lactate was reduced significantly with theoverexpression of the new FDH.

In addition, the chemostat results suggest that the new FDH is able tocompete very effectively with the native FDH; therefore, it is notnecessary to eliminate the native FDH activity in order to achieve thedesired results, making this approach easier to implement in a varietyof applications. It should also be noted that the effect of this systemwas reduced under the current experimental conditions as compared to theuncontrolled anaerobic tube experiments reported previously, in whichthe Et/Ac ratio represented a 27-fold increase with substitution of thenative by the NAD+-dependent FDH (see FIG. 4B).

Thus, the data demonstrate that NADH manipulations in a systemcomprising a NADH recycling system achieve redirection of carbon fluxesto produce reduced products. Based on this data, effects on otherreduced cofactors such as FADH or NADPH directly are expected because ofinterconversions among the reduced cofactors in the cell. This reasoningleads to a plausible application of the present invention in terms ofmanipulating intracellular availability of other reduced cofactors suchas FADH, a flavin coenzyme that is usually tightly bound to oneparticular enzyme, and NADPH, a nicotinamide cofactor that like NADHacts as a hydrogen carrier and is capable of diffusing from enzyme toenzyme.

Example 18 NADH Recycling in Biodesulfurization

The usual model for the study of biodesulfurization is the compounddibenzothiophene. It has been extensively studied in the context ofnonbiological and biological desulfurization. Dibenzothiophene is amember of a class of polyaromatic sulfur heterocycles (PASHs), and oneof thousands of PASHs found in a hydrotreated diesel sample. Alkylateddibenzothiophenes are also target molecules for biodesulfurizationtechnology.

Cells capable of biodesulfurization are transformed with a recombinantNADH recycling system.

Known bacterial strains which are capable of breaking downdibenzothiophene using this pathway include Rhodococcus strains IGTS8,T09, and RA-18, and Gordonia desulfuricans 213E. Also capable ofbiodesulfurization are E. coli that express recombinant genes fromRhodococcus, and Pseudomonas putida that express recombinant genes fromRhodococcus. Gordonia rubropertinctus strain T08 is capable ofbiodesulfurization using a novel pathway.

The first step in the desulfurization pathway is the transfer of thetarget molecules from oil into the cells. Rhodococcus sp. and otherbacteria have been shown to metabolize many insoluble molecules throughdirect transfer from oil into the cells.

Dibenzothiophene monooxygenase (SEQ ID NO:12, Accession NO: P54995), theenzyme responsible for the first two oxidation in the biodesulfurizationpathway has been isolated and characterized, and its gene has beencloned and sequenced. The enzyme catalyzes the transfer of an electronfrom flavin mononucleotide to dibenzothiophene, and catalyzes theoxidation of dibenzothiophene to the sulfoxide and the oxidation of thesulfoxide to the sulfone. The cleavage of the first carbon-sulfurlinkage of dibenzothiophene is catalyzed by dibenzothiophene sulfonemonooxygenase (SEQ ID NO:13, Accession NO: P54997). This enzyme and itsgene have been characterized. Production of sulfite is the last reactionin the pathway. This is catalyzed by a desulfinase (SEQ ID NO:14,Accession NO: P54998), whose gene has been cloned and sequenced. Sulfiteis released as well as an oil soluble product, hydroxyl biphenyl.

NADH is required in this reaction system to keep the supply of reducedflavin mononucleotide in balance.

Additionally, large-scale biodesulfurization in bacteria utilizingrecombinant, constitutively-expressed members of biodesulfurizationpathway (dsz class genes), requires NADH, which can be limiting.

Example 19 NADH Recycling in Biopolymer Production

Polyhydroxyalkanoates (PHAs) are linear polyesters produced in nature inbacteria. Bacteria accumulate PHAs when a carbon source is abundant. Thegenes involved in PHA synthesis from well over 20 differentmicroorganisms have been characterized. These recombinant genes aretransformed into cells comprising the NADH recycling system. The genesinvolved in PHA synthesis include beta-ketothiolase,acetoacetyl-CoA-reductase, butyrate dehydrogenase andpoly-3-hydroxybutyrate synthase.

Bacterial cells capable of PHA synthesis include the carbon monoxide(CO)-resistant strain of the hydrogen bacteria Ralstonia eutropha B5786,Synechocystis sp. PCC6803, and Pseudomonas corrugata. These bacteria aretransformed with a recombinant NADH recycling system.

NADH recycling allows increased polymer production.

Example 20 NADH Recycling in Polypeptide Production

Cells comprising the NADH recycling system are transformed with a vectorpSM552-545C-, containing the lacZ gene, which encodesbeta-galactosidase. The expression of the lacZ gene is regulated by apowerful pH-inducible promoter. Experiments are conducted in awell-controlled fermenter under optimal conditions for the particularexpression system. The expression of the lacZ gene is induced bychanging the pH from 7.5, which has minimal induction, to a pH of 6.0,which is the optimal induction pH. NADH, acetate, and beta-galactosidaseproduction are monitored through standard means in the art. Increasedbeta-galactosidase production is associated with lower levels ofacetate. Lower levels of acetate production are associated with cellscomprising the NADH recycling system.

REFERENCES

All patents and publications mentioned in the specification areindicative of the level of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

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U.S. Pat. No. 6,001,590

U.S. Pat. No. 5,264,092

U.S. Pat. No. 5,520,786

U.S. Pat. No. 5,393,615

U.S. Pat. No. 4,683,202

U.S. Pat. No. 5,928,906

U.S. Pat. No. 5,925,565

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EP 266032

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One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objectives and obtain the ends andadvantages mentioned as well as those inherent therein. Systems,pharmaceutical compositions, treatments, methods, procedures andtechniques described herein are presently representative of thepreferred embodiments and are intended to be exemplary and are notintended as limitations of the scope. Changes therein and other useswill occur to those skilled in the art which are encompassed within thespirit of the invention or defined by the scope of the pending claims.

1-10. (canceled)
 11. A cell comprising a recombinant NADH-recyclingsystem, wherein said system comprises a heterologous nucleotide sequenceencoding an NAH+-dependent dehydrogenase.
 12. The cell of claim 11,wherein said heterologous nucleotide sequence encoding an NAH+-dependentdehydrogenase is a nucleotide sequence encoding an NAH+-dependentformate dehydrogenase.
 13. The cell of claim 11, wherein saidheterologous nucleotide sequence encoding an NAH+-dependentdehydrogenase is a yeast nucleotide sequence encoding an NAH+-dependentdehydrogenase.
 14. The cell of claim 11, wherein said heterologousnucleotide sequence encoding an NAH+-dependent dehydrogenase is a yeastnucleotide sequence encoding an NAH+-dependent formate dehydrogenase.15. The cell of claim 11, wherein said heterologous nucleotide sequenceencoding an NAH+-dependent dehydrogenase is a nucleotide sequence fromCandida boidinii.
 16. The cell of claim 11, wherein said heterologousnucleotide sequence encoding an NAH+-dependent dehydrogenase is anucleotide sequence from Candida boidinii comprising SEQ ID NO:
 1. 17.The cell of claim 11, wherein said cell is a bacterium.
 18. The cell ofclaim 11, wherein said cell is E. coli.
 19. The cell of claim 11,wherein said cell is a member of the genus Rhodococcus. 20-47.(canceled)